Migraine is one of the most common neurovascular disorders with noticeable genetic predisposition. Understanding the mechanisms of migraine will lead to more specific treatments. Serotonin (5-HT) is an extracellular signaling molecule with a multitude of functions in the central nervous system (CNS) and in the periphery. There is a significant body of evidence implicating that migraine may result from a chronic low 5-HT disposition. Nevertheless, the causal relationship between abnormal 5-HT level and the manifestation of migraine headache has not been firmly established. Even less is known about the contribution of central versus peripheral 5-HT to migraine pathophysiology. The Lmx1b conditional knock-out mice (Lmx1bf/f/p) offers a unique opportunity to address these questions. Adult Lmx1bf/f/p mice lack almost all the central serotonergic neurons in the brainstem, whereas 5-HT expression in the periphery is not affected. In this project we propose to use the Lmx1bf/f/p mice in combination with molecular and pharmacological approaches to explore the contribution of 5-HT system to migraine pathophysiology, emphasizing on elucidating the role of central versus peripheral 5-HT in the generation of migraine headache. We hypothesize that chronic low 5-HT state result in the hyperexcitation of: 1) the brain areas that respond to migraine triggers; 2) the trigeminovascular pathway subserving head pain or 3) both; thereby increasing the susceptibility to migraine headache. In the first aim we will investigate whether the Lmx1bf/f/p mice exhibit increased susceptibility to cortical spreading depression (CSD) - a stimulus that mimic migraine trigger. The CSD properties will be used as a marker to monitor cortical excitability. The second aim of the proposal addresses whether the lack of central 5-HT neurons affects the excitability of the trigeminovascular pathway per se. The pattern of Fos protein expression in cervical/medullary dorsal horn will be used to monitor the activity of the trigeminal nociceptive pathway. Finally, we will pharmacologically deplete the peripheral 5-HT in Lmx1bf/f/p mice to see if this affects the excitability of the 'migraine circuit'. And if so, whether the effects of central and peripheral 5-HT on migraine circuit is additive, synergistic or antagonistic. Together, these experiments will offer valuable insights into the contribution of central and peripheral 5-HT system to migraine pathophysiology. The work proposed here will set a stage that allows us to further advance our understanding of the migraine pathophysiology as well as progression. This will greatly facilitate drug development for both preventive and palliative therapies of migraine.